Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a phytoestrogenic component of Rheum plant extracts which has been used for medical treatment since ancient times. It has been shown to have anti-inflammatory and anti-cancer effects. In our research, we aimed to study the biological effects of emodin on MCF-7 and MDA-231 cell lines. Cytotoxicity assays showed that emodin treatment for 48hours caused a concentration dependent decrease in viable cell numbers of both cell lines. As determined by cell counting with tryphan blue, IC50 values were 8.40 and 12.17 &micro / g/ml for MCF-7 and MDA-231 cells, respectively. Apoptotic effects of emodin was investigated by measuring the changes in apoptotic and antiapoptotic gene expressions by qRT-PCR. In MCF-7 cells, Bax expression increased with increasing emodin concentrations, while Bcl-2 expression was downregulated. Bax/Bcl-2 ratio was calculated as 9.2 fold at 10&micro / g emodin/ml treatment for 48 hours, indicating stimulation of apoptosis. However, Bax/Bcl-2 ratio was found 1.6 fold for MDA-231 cells. These results were in accordance with the results obtained from microarray analysis of related gene expressions. MCF-7 cells were more apoptotic in comparison to MDA-231 cells. DNA fragmentation was observed in MCF-7 cells by TUNEL method. GST enzyme activity that was measured using CDNB as substrate, was increased 100% with respect to control up to 5&micro / g emodin/ml treatment of MCF-7 cells for 48 hours. However, effect of emodin on GST activity in MDA-231 cells was found insignificant. According to microarray analysis results, emodin affected the gene expressions of GST isozymes in MCF-7 cells much more than in MDA-231 cells.

ABSTRACT TRANSFORMATION OF POTATO WITH MYB4 TRANSCRIPTION FACTOR AND EVALUATION OF ABIOTIC STRESS TOLERANCE AND GENE EXPRESSION PROFILES IN TRANSGENIC PLANTS Kalemtas, G&uuml / ls&uuml / m Ph.D., Department of Biology Supervisor: Prof. Dr. H&uuml / seyin Avni &Ouml / ktem February 2011, 257 pages Potato (Solanum tuberosum L. cv. Kennebec) was transformed via Agrobacterium tumefaciens (EHA105) harbouring two different binary vectors containing Oryza sativa myb4 gene, which encodes MYB4 transcription factor / under the control of CaMV35S promoter or cold inducible COR15a promoter. The transgenic plants were not growth retarded and there was no significant difference (p&lt / 0.05) in their tuber yield compared to wild-type plants. Wild-type and transgenic plants were subjected to abiotic stresses to compare their stress tolerances. There was no significant difference in boron, freezing and drought tolerances of wild-type and transgenic lines. Two of the transgenic lines were more salt tolerant than wild-type with respect to growth parameters. Transcriptomes of wild-type and these two lines, one expressing myb4 under the control of 35S promoter and the other COR15a promoter, were analyzed to elucidate the myb4-regulated processes and downstream target genes in potato. Differentially regulated genes in transgenic lines showed that myb4 controls a large and complex transcriptional network associated with diverse cellular processes, primarily defense and rescue, metabolism and development. Genes involved in sucrose synthesis, some peroxidases and CBF3 transcription factor were up-regulated in transgenic plants upon exposure to freezing stress. This suggested that myb4 may configure freezing response in potato primarily by oxidative stress defence mechanisms, osmotic adjustment or activation of CBF3 regulated genes that may confer cold tolerance. Despite up-regulation of these stress related genes, transgenic potato was not more drought or freezing tolerant compared to WT under the tested conditions. Further experiments should be conducted to better elucidate the involvement of these genes in regulation of stress response in transgenic potato expressing myb4.

Wheat is the most important grain crop grown in our country providing greatest part of the daily nutritional requirement. Abiotic factors including salinity, drought, cold and heat stresses affect quality and yield of wheat varieties used for the production of both bread and pasta flour. NAC proteins form one of the widest families of plant specific transcription factors. Members of this family are related with development, defense and abiotic stress responses. TaNAC69-1 and TtNAM-B2 genes were isolated from T.aestivum and T.turgidum, respectively. Then they were cloned into different monocot and dicot expression vectors to be used for further wheat and tobacco genetic transformation studies. To understand effects of salinity, drought, cold and heat stresses on expression profiles of TaNAC69-1 and TtNAM-B2 genes, quantitative real time PCR was performed. The time series expression profiles of TaNAC69-1 show that it was signi

Microarray technology enables researchers to measure the expression levels of thousands of genes simultaneously to understand relationships between genes, extract pathways, and in general understand a diverse amount of biological processes such as diseases and cell cycles. While microarrays provide the great opportunity of revealing information about biological processes, it is a challenging task to mine the huge amount of information contained in the microarray datasets. Generally, since an accurate model for the data is missing, first a clustering algorithm is applied and then the resulting clusters are examined manually to find genes that are related with the biological process under inspection. We need automated methods for this analysis which can be used to eliminate unrelated genes from data and mine for biologically important genes. Here, we introduce a general methodology which makes use of traditional clustering algorithms and involves integration of the two main sources of biological information, Gene Ontology and interaction networks, with microarray data for eliminating unrelated information and find a clustering result containing only genes related with a given biological process. We applied our methodology successfully on a number of different cases and on different organisms. We assessed the results with Gene Set Enrichment Analysis method and showed that our final clusters are highly enriched. We also analyzed the results manually and found that most of the genes that are in the final clusters are actually related with the biological process under inspection.

In the present investigation, microarray analysis was used to identify potential TCDD gene targets. Three microarray experiments were performed to study the effect of TCDD in an established chicken B-cell line (DT40), in a chicken macrophage cell line (HD11), and in the bursa of Fabricius from embryos exposed in ovo at 6 days of incubation. From the DT40 microarray analyses, clones with sequence similarity to the apoptotic genes caspase 8 and caspase 9, and the transcription factor NFΜB, among others, were identified. Real-time quantitative polymerase chain reaction (RT-PCR) revealed that TCDD elicits aryl hydrocarbon receptor (AhR)-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3 (see chapter III). During the course of the HD11 microarray analyses, a consistent down-regulation of the matrix metalloprotease MMP-2 was observed. This finding was the basis for the hypothesis that TCDD has an effect on the gene expression of the MMP-2 and MMP-9 in macrophages. Then, gene expression analysis and functional zymography showed that TCDD impairs the MMP-2 and MMP-9 response to LPS stimulation in HD11 chicken macrophages (see chapter V). The microarray analyses of the embryonic bursa of Fabricius provided the basis to further study of the effect of TCDD in the chicken embryo. The shifted genes were classified according to their function. The down-regulated genes included: precursor of matrix metalloprotease-inhibitor, histone acyl-transferase 1, homeobox protein CUX-2, Death Associated Protein Kinase, and UDPglucosyl transferase, among others. The up-regulated genes included: phosphoinositidespecific phospholipase, acyl Co-A oxidase, and protein effector of Cdc42, among others. Together, these microarray analyses produced a database of genes of interest that will provide sufficient hypotheses to inspire multiple investigations aimed at confirming and refining the gene expression alterations as a consequence of TCDD exposure.

Leukocyte transmigration is a critical step of the inflammatory process. In this project I have examined leukocyte responses to transmigration and endothelial responses to both chemical and mechanical stimuli which are known to be involved in leukocyte transmigration. My work has identified ~2500 differentially expressed genes following endothelial exposure to interleukin-1 beta (IL1β). Interestingly, IL1β induces up-regulation of claudin-1 and pre-b-cell colony enhancing factor and down-regulation of claudin-5 and occludin, which are all involved in maintaining endothelial cell-cell junctions. Analysis of endothelial cell (EC) transcriptional changes following neutrophil transmigration found few differentially expressed genes in comparison to IL1β treated ECs; indicating that the effects of transmigration on ECs are minimal in comparison to the global transcriptional changes induced by IL1β. Atherosclerosis, characterized by monocyte accumulation within the vessel lumen, is found in regions of flow reversal and low time averaged oscillatory shear stress. I have examined the effects of this type of shear stress on endothelial cell gene expression. My data indicates that most genes differentially expressed under these conditions are controlled by low average shear stress rather than flow reversal. These differentially expressed genes are involved in regulating the cell cycle and the immune response. My work shows that cell proliferation is increased following exposure to low steady shear stress or exposure to reversing oscillatory flow in comparison to high steady shear stress. Additionally monocyte adhesion is increased following exposure of ECs to reversing oscillatory flow. My work has also examined the impact of transmigration on monocyte gene expression. I have identified genes which are differentially expressed in monocytes by exposure to EC secretions, monocyte/EC contact, and diapedesis. I have also shown that freshly isolated human monocytes have reduced apoptosis following transmigration. Surprisingly, I also found that monocytes had reduced expression of anti-microbial peptides following transmigration. Overall my work identifies important endothelial and leukocyte transcriptional responses to the process of transmigration which extends from cytokine stimulation through diapedesis.

<p>Today’s rapid development of powerful tools for geneexpression analysis provides unprecedented resources forelucidating complex molecular events.</p><p>The objective of this workhas been to apply, combine andevaluate tools for analysis of differential gene expressionusing atherosclerosis as a model system. First, an optimisedsolid-phase protocol for representational difference analysis(RDA) was applied to two<i>in vitro</i>model systems. Initially, The RDA enrichmentprocedure was investigated by shotgun cloning and sequencing ofsuccessive difference products. In the subsequent steps,combinations of RDA and microarray analysis were used tocombine the selectivity and sensitivity of RDA with thehigh-throughput nature of microarrays. This was achieved byimmobilization of RDA clones onto microarrays dedicated forgene expression analysis in atherosclerosis as well ashybridisation of labelled RDA products onto global microarrayscontaining more than 32,000 human clones. Finally, RDA wasapplied for the investigation of the focal localisation ofatherosclerotic plaques in mice using<i>in vivo</i>tissue samples as starting material.</p><p>A large number of differentially expressed clones wereisolated and confirmed by real time PCR. A very diverse rangeof gene fragments was identified in the RDA products especiallywhen they were screened with global microarrays. However, themicroarray data also seem to contain some noise which is ageneral problem using microarrays and should be compensated forby careful verification of the results.</p><p>Quite a large number of candidate genes related to theatherosclerotic process were found by these studies. Inparticular several nuclear receptors with altered expression inresponse to oxidized LDL were identified and deserve furtherinvestigation. Extended functional annotation does not liewithin the scope of this thesis but raw data in the form ofnovel sequences and accession numbers of known sequences havebeen made publicly available in GenBank. Parts of the data arealso available for interactive exploration on-line through aninteractive software tool. The data generated thus constitute abase for new hypotheses to be tested in the field ofatherosclerosis.</p><p><b>Keywords:</b>representational difference analysis, geneexpression profiling, microarray analysis, atherosclerosis,foam cell formation</p>

Source: Masters Abstracts International, Volume: 47-06, page: 3373. Adviser: Wenyaw Chan. Includes bibliographical references.

High throughput technologies such as microarrays and modern genome sequencers produce enormous amounts of data that require novel data processing. This thesis proposes a method called Interdependent Correlation Cluster (ICC) to analyze the relations between genes represented by microarray data that are conditioned on a specific target gene. Based on Correlation Clustering, the proposed method analyzes a large set of correlation values related to the gene expression profiles extracted from given microarray datasets. The proposed method works on any size microarray datasets and could be applied to any target gene. In this study the selected target gene, NSF1 /USV1 / YPL230W, encodes a poorly characterized C2H2 zinc finger transcription factor (TF) involved in stress responses in yeast. The method is successful in the identification of novel NSF1 functional roles during fermentation stress conditions in the M2 industrial yeast strain. The new identified functions include regulation of energy and sulfur metabolism, protein synthesis, ribosomal assembly and protein trafficking as well as other processes. NSF1 involvement in sulfur metabolism was experimentally confirmed using biological laboratory techniques. Importantly, implication of NSF1 in sulfur metabolism regulation has highly relevant implications to wine and beer production industries concerned with production of compounds having sulfur-like off odour (SLO) and toxic properties. The correlation clustering also provides a means of understanding complex interactions existing between genes. / The pdf file contains numerous hyperlinks and bookmarks to facilitate navigation. This thesis will be of interest to those working with topics such as data mining of microarray data, novel gene function discovery and prediction, and genome-wide responses to fermentation stresses. / Ministry of Training, Colleges and Universities of Ontario (Ontario Graduate Scholarship and Ontario Graduate Scholarships in Science and Technology); The Natural Sciences and Engineering Research Council of Canada (NSERC)

The development of microarray technology has provided an unprecedented wealth of data on gene expression in various tissue and cell types. Few studies have, however, taken full advantage of these data by selecting and then extensively characterising the functions of particular genes chosen from these microarray datasets. In this study, after analysing differentially-regulated genes revealed by microarray analysis of human mast cells activated via Fc??RI cross-linking, we chose two promising gene candidates for further research, A20 and Gem. Our group??s extensive gene expression database of major leukocytes showed that both A20 and Gem were up-regulated in other leukocyte types, and yet neither of these genes has been extensively explored in mast cells or in the immune system prior to our study. In order to investigate the first of these genes selected for further study, A20, we utilised both A20-deficient mast cells and mast cells in which A20 was over-expressed. Our findings establish for the first time that A20 is an important regulator of mast cell inflammatory responses to both LPS and Fc??RI cross-linking, and that it plays a novel role in mast cell proliferation. Our study of the second gene chosen for investigation, Gem, was conducted in a Gemdeficient mouse model developed by our group. In this study, we investigated the effect of Gem deficiency in two key immune cell types, macrophages and T-cells, complementing the work of a previous group member who investigated Gem deficiency in mast cells. Our results clearly exclude a role for Gem in macrophage and T-cell effector responses, and further establish that Gem is dispensable for in vivo inflammatory responses in models of delayed-type hypersensitivity and allergic airway inflammation. In addition to these findings, and given that the physiological role of Gem was not yet understood prior to our study, we extended our investigation to explore a potential function for Gem in the metabolic system. Using Gem-deficient mice, we found that Gem is necessary for insulin secretion from pancreatic islets. These findings confirm the potential for microarray expression data to reveal excellent gene candidates for further research and functional characterisation.